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BioRxiv : the Preprint Server For... Jun 2024Gangliosides are sialylated glycosphingolipids with essential but enigmatic functions in healthy and disease brains. GD3 is the predominant species in neural stem cells...
Gangliosides are sialylated glycosphingolipids with essential but enigmatic functions in healthy and disease brains. GD3 is the predominant species in neural stem cells (NSCs) and GD3-synthase (sialyltransferase II; ) knockout (GD3S-KO) revealed reduction of postnatal NSC pools with severe behavioral deficits including cognitive impairment, depression-like phenotypes, and olfactory dysfunction. Exogenous administration of GD3 significantly restored the NSC pools and enhanced the stemness of NSCs with multipotency and self-renewal, followed by restored neuronal functions. Our group discovered that GD3 is involved in the maintenance of NSC fate determination by interacting with epidermal growth factor receptors (EGFRs), by modulating expression of cyclin-dependent kinase (CDK) inhibitors p27 and p21, and by regulating mitochondrial dynamics via associating a mitochondrial fission protein, the dynamin-related protein-1 (Drp1). Furthermore, we discovered that nuclear GM1 promotes neuronal differentiation by an epigenetic regulatory mechanism. GM1 binds with acetylated histones on the promoter of as well as on the in differentiated neurons. In addition, epigenetic activation of the GM2S gene was detected as accompanied by an apparent induction of neuronal differentiation in NSCs responding to an exogenous supplement of GM1. Interestingly, GM1 induced epigenetic activation of the gene, with recruitment of Nurr1 and PITX3, dopaminergic neuron-associated transcription factors, to the promoter region. In this way, GM1 epigenetically regulates dopaminergic neuron specific gene expression, and it would modify Parkinson's disease. Multifunctional gangliosides significantly modulate lipid microdomains to regulate functions of important molecules on multiple sites: the plasma membrane, mitochondrial membrane, and nuclear membrane. Versatile gangliosides regulate functional neurons as well as sustain NSC functions via modulating protein and gene activities on ganglioside microdomains. Maintaining proper ganglioside microdomains benefits healthy neuronal development and millions of senior citizens with neurodegenerative diseases. Here, we introduce how to isolate GD3 and GM1 and how to administer them into the mouse brain to investigate their functions on NSC fate determination and nerve cell specification.
PubMed: 38915682
DOI: 10.1101/2024.06.09.598109 -
International Journal of Molecular... Jun 2024Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes...
Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.
Topics: Humans; Autophagy; Extracellular Vesicles; Cell Line, Tumor; Membrane Microdomains; Exosomes; Tetraspanin 30; Fibrosarcoma; Microtubule-Associated Proteins
PubMed: 38892363
DOI: 10.3390/ijms25116175 -
Cells May 2024Podocyte health is vital for maintaining proper glomerular filtration in the kidney. Interdigitating foot processes from podocytes form slit diaphragms which regulate... (Review)
Review
Podocyte health is vital for maintaining proper glomerular filtration in the kidney. Interdigitating foot processes from podocytes form slit diaphragms which regulate the filtration of molecules through size and charge selectivity. The abundance of lipid rafts, which are ordered membrane domains rich in cholesterol and sphingolipids, near the slit diaphragm highlights the importance of lipid metabolism in podocyte health. Emerging research shows the importance of sphingolipid metabolism to podocyte health through structural and signaling roles. Dysregulation in sphingolipid metabolism has been shown to cause podocyte injury and drive glomerular disease progression. In this review, we discuss the structure and metabolism of sphingolipids, as well as their role in proper podocyte function and how alterations in sphingolipid metabolism contributes to podocyte injury and drives glomerular disease progression.
Topics: Podocytes; Sphingolipids; Humans; Animals; Lipid Metabolism; Kidney Diseases; Membrane Microdomains
PubMed: 38891023
DOI: 10.3390/cells13110890 -
Microbiology Spectrum Jun 2024The phylum Oomycota contains economically important pathogens of animals and plants, including , the causal agent of the fish disease saprolegniasis. Due to intense fish...
The phylum Oomycota contains economically important pathogens of animals and plants, including , the causal agent of the fish disease saprolegniasis. Due to intense fish farming and banning of the most effective control measures, saprolegniasis has re-emerged as a major challenge for the aquaculture industry. Oomycete cells are surrounded by a polysaccharide-rich cell wall matrix that, in addition to being essential for cell growth, also functions as a protective "armor." Consequently, the enzymes responsible for cell wall synthesis provide potential targets for disease control. Oomycete cell wall biosynthetic enzymes are predicted to be plasma membrane proteins. To identify these proteins, we applied a quantitative (iTRAQ) mass spectrometry-based proteomics approach to the plasma membrane of the hyphal cells of , providing the first complete plasma membrane proteome of an oomycete species. Of significance is the identification of 65 proteins enriched in detergent-resistant microdomains (DRMs). analysis showed that DRM-enriched proteins are mainly involved in molecular transport and β-1,3-glucan synthesis, potentially contributing to pathogenesis. Moreover, biochemical characterization of the glycosyltransferase activity in these microdomains further supported their role in β-1,3-glucan synthesis. Altogether, the knowledge gained in this study provides a basis for developing disease control measures targeting specific plasma membrane proteins in .IMPORTANCEThe significance of this research lies in its potential to combat saprolegniasis, a detrimental fish disease, which has resurged due to intensive fish farming and regulatory restrictions. By targeting enzymes responsible for cell wall synthesis in , this study uncovers potential avenues for disease control. Particularly noteworthy is the identification of several proteins enriched in membrane microdomains, offering insights into molecular mechanisms potentially involved in pathogenesis. Understanding the role of these proteins provides a foundation for developing targeted disease control measures. Overall, this research holds promise for safeguarding the aquaculture industry against the challenges posed by saprolegniasis.
PubMed: 38888349
DOI: 10.1128/spectrum.00348-24 -
Journal of Structural Biology: X Jun 2024Physical properties of biological membranes directly or indirectly govern biological processes. Yet, the interplay between membrane and integral membrane proteins is...
Physical properties of biological membranes directly or indirectly govern biological processes. Yet, the interplay between membrane and integral membrane proteins is difficult to assess due to reciprocal effects between membrane proteins, individual lipids, and membrane architecture. Using solid-state NMR (SSNMR) we previously showed that KirBac1.1, a bacterial Inward-Rectifier K channel, nucleates bilayer ordering and microdomain formation through tethering anionic lipids. Conversely, these lipids cooperatively bind cationic residues to activate the channel and initiate K flux. The mechanistic details governing the relationship between cooperative lipid loading and bilayer ordering are, however, unknown. To investigate, we generated KirBac1.1 samples with different concentrations of C-lableded phosphatidyl glycerol (PG) lipids and acquired a full suite of SSNMR 1D temperature series experiments using the ordered all-trans (AT) and disordered -gauche (TG) acyl conformations as markers of bilayer dynamics. We observed increased AT ordered signal, decreased TG disordered signal, and increased bilayer melting temperature with increased PG concentration. Further, we identified cooperativity between ordering and direct binding of PG lipids, indicating KirBac1.1-driven bilayer ordering and microdomain formation is a classically cooperative Hill-type process driven by and predicated upon direct binding of PG lipids. Our results provide unique mechanistic insight into how proteins and lipids in tandem contribute to supramolecular bilayer heterogeneity in the lipid membrane.
PubMed: 38883399
DOI: 10.1016/j.yjsbx.2024.100101 -
Scientific Reports Jun 2024Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral...
Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral heterogeneity of the lipid bilayer and associated clustering of distinct membrane proteins contribute to the spatial organization of numerous cellular processes. Here, we show that membrane microdomains within the endoplasmic reticulum (ER) of yeast cells are reorganized during metabolic reprogramming and aging. Using biosensors with varying transmembrane domain length to map lipid bilayer thickness, we demonstrate that in young cells, microdomains of increased thickness mainly exist within the nuclear ER, while progressing cellular age drives the formation of numerous microdomains specifically in the cortical ER. Partitioning of biosensors with long transmembrane domains into these microdomains increased protein stability and prevented autophagic removal. In contrast, reporters with short transmembrane domains progressively accumulated at the membrane contact site between the nuclear ER and the vacuole, the so-called nucleus-vacuole junction (NVJ), and were subjected to turnover via selective microautophagy occurring specifically at these sites. Reporters with long transmembrane domains were excluded from the NVJ. Our data reveal age-dependent rearrangement of the lateral organization of the ER and establish transmembrane domain length as a determinant of membrane contact site localization and autophagic degradation.
Topics: Endoplasmic Reticulum; Autophagy; Saccharomyces cerevisiae; Membrane Microdomains; Cellular Senescence; Saccharomyces cerevisiae Proteins; Vacuoles; Membrane Proteins
PubMed: 38871812
DOI: 10.1038/s41598-024-64493-8 -
Biophysical Journal Jun 2024Annexin A2 (A2)-induced microdomain formation is a key step in biological processes such as Ca-mediated exocytosis in neuroendocrine cells. In this work, a total of 15...
Annexin A2 (A2)-induced microdomain formation is a key step in biological processes such as Ca-mediated exocytosis in neuroendocrine cells. In this work, a total of 15 coarse-grained molecular dynamics simulations were performed on vesicle models having a diameter of approximately 250 Å for 15 μs each using the Martini2 force field. Five simulations were performed in the presence of 10 A2, 5 in the presence of A2 but absence of PIP, and 5 simulations in the absence of A2 but presence of PIP. Consistent results were generated among the simulations. A2-induced PIP microdomain formation was observed and shown to occur in three phases: A2-vesicle association, localized A2-induced PIP clustering, and A2 aggregation driving PIP microdomain formation. The relationship between A2 aggregation and PIP microdomain formation was quantitatively described using a novel method which calculated the variance among protein and lipid positions via the Fréchet mean. A large reduction in PIP variance was observed in the presence of A2 but not in its absence. This reduction in PIP variance was proportional to the reduction observed in A2 variance and demonstrates that the observed PIP microdomain formation is dependent upon A2 aggregation. The three-phase model of A2-induced microdomain formation generated in this work will serve as a valuable guide for further experimental studies and the development of novel A2 inhibitors. No microdomain formation was observed in the absence of A2 and minimal A2-membrane interaction was observed in the absence of PIP.
PubMed: 38859585
DOI: 10.1016/j.bpj.2024.06.006 -
BioRxiv : the Preprint Server For... May 2024Fungal plasma membrane proteins represent key therapeutic targets for antifungal agents, yet their structure and spatial distribution in the native context remain poorly...
Fungal plasma membrane proteins represent key therapeutic targets for antifungal agents, yet their structure and spatial distribution in the native context remain poorly characterized. Herein, we employ an integrative multimodal approach to elucidate the structural and functional organization of plasma membrane protein complexes in , focusing on prominent and essential membrane proteins, the polysaccharide synthase β-(1,3)-glucan synthase (GS) and the proton pump Pma1. Cryo-electron tomography (cryo-ET) and live cell imaging reveal that GS and Pma1 are heterogeneously distributed into distinct plasma membrane microdomains. Treatment with caspofungin, an echinocandin antifungal that targets GS, alters the plasma membrane and disrupts the native distribution of GS and Pma1. Based on these findings, we propose a model for echinocandin action that considers how drug interactions with the plasma membrane environment lead to inhibition of GS. Our work underscores the importance of interrogating the structural and dynamic characteristics of fungal plasma membrane proteins to understand function and facilitate precisely targeted development of novel antifungal therapies.
PubMed: 38854035
DOI: 10.1101/2024.05.29.596243 -
Plant Communications Jun 2024The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the...
The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the nodule, an organ in which mutualistic symbiosis with the N-fixing rhizobia occurs. A mature soybean root nodule is characterized by a central infection zone where the atmospheric nitrogen is fixed and assimilated by the symbiont, resulting from the close cooperation between the plant cell and the bacteria. To date, the transcriptome of individual cells isolated from developing soybean nodules has been established, but the transcriptomic signatures of the cells of the mature soybean nodule have not yet been characterized. Applying single nucleus RNA-seq and Molecular Cartography technologies, we precisely characterized the transcriptomic signature of the soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B. diazoefficiens-infected cells in the mature soybean nodule including those actively involved in nitrogen fixation, and those engaged in senescence. The mining of the single cell-resolution nodule transcriptome atlas and associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes controlling the nodulation process. For instance, we functionally characterized the role of GmFWL3, a plasma membrane microdomain-associated protein controlling rhizobia infection. Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule.
PubMed: 38845198
DOI: 10.1016/j.xplc.2024.100984 -
ELife Jun 2024The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these...
The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, etention sing elective ooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.
Topics: Protein Transport; Endoplasmic Reticulum; Golgi Apparatus; Membrane Microdomains; Secretory Pathway; Humans; Kinetics; Cell Membrane; Membrane Proteins; HeLa Cells
PubMed: 38837189
DOI: 10.7554/eLife.89306